Diammonium Glycyrrhizinate Protects against Non-Alcoholic Fatty

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Diammonium Glycyrrhizinate Protects against NonAlcoholic Fatty Liver Disease in mice through modulation of gut microbiota and restoring intestinal barrier Yun Li, Tianyu Liu, Chen Yan, Runxiang Xie, Zixuan Guo, Sinan Wang, Yujie Zhang, Zhengxiang Li, Bangmao Wang, and Hailong Cao Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00347 • Publication Date (Web): 23 Jul 2018 Downloaded from http://pubs.acs.org on July 24, 2018

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Molecular Pharmaceutics

Diammonium Glycyrrhizinate Protects against Non-Alcoholic Fatty Liver Disease in mice through modulation of gut microbiota and restoring intestinal barrier Yun Li1, 2+, Tianyu Liu1+, Chen Yan2, Runxiang Xie1, Zixuan Guo1, Sinan Wang1, Yujie Zhang3, Zhengxiang Li2, Bangmao Wang1, Hailong Cao1∗ 1

Department of Gastroenterology and Hepatology, General Hospital, Tianjin Medical University, Tianjin 300052, China

2

Department of Pharmacy, General Hospital, Tianjin Medical University, Tianjin 300052,

China 3

Department of Pathology, General Hospital, Tianjin Medical University, Tianjin 300052,

China

+ The First two authors contributed equally to this work. ∗ Corresponding author: Hailong Cao, Department of Gastroenterology and Hepatology, General Hospital, Tianjin Medical University, Tianjin 300052, China. E–mail: [email protected]

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ABSTRACT Nonalcoholic fatty liver disease (NAFLD), as a common chronic liver disorder, is prevalent in the world. Recent evidence demonstrates that the “gut-liver axis” is related well to the progression of NAFLD, which regards gut microbiota and intestinal barrier as two critical factors correlated with NAFLD. Diammonium glycyrrhizinate (DG), a compound of natural bioactive pentacyclic triterpenoid glycoside, is the main component of licorice roots extracts. The anti-inflammatory and liver protection effects of DG have already been reported, but to date, the mechanism has not been fully elucidated. In this research, we observed that DG reduced body weight, liver steatosis, as well as hepatic inflammatory in NAFLD model mice induced by high fat diet. Illumina sequencing of the 16S rRNA revealed that DG intervention notably altered the composition of the gut microbiota in NAFLD mice. The richness of gut microbiota was significantly increased by DG. Specifically, DG reduced the Firmicutes-to-Bacteroidetes ratio and the endotoxin-producing bacteria such as Desulfovibrio, and elevated the abundance of probiotics such as Proteobacteria and Lactobacillus. Interestingly DG could augment the levels of short-chain fatty acids (SCFAs)-producing bacteria such as Ruminococcaceae and Lachnospiraceae and promote SCFAs production. In addition, DG supplementation dramatically alleviated the intestinal low-grade inflammation. Meanwhile, DG improved the expression of tight junction proteins, the goblet cells number and mucin secretion, and sequentially enhanced the function of intestinal barrier. Collectively, the prevention of NAFLD by DG might be mediated by modulating gut microbiota and restoring intestinal barrier. KEYWORDS: Nonalcoholic fatty liver disease, diammonium glycyrrhizinate, gut microbiota, intestinal barrier


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INTRODUCTION Nonalcoholic fatty liver disease (NAFLD) is increasingly considered to be a hepatic manifestation of metabolic syndrome featured by insulin resistance and systemic complex.1-3 And NAFLD represents a spectrum of hepatic pathology ranging from the sole presence of hepatic steatosis (non-alcoholic fatty liver, NAFL) to the development of steatosis in combination with inflammation, fibrosis (non-alcoholic steatohepatitis, NASH) or cirrhosis.4 It has been proposed that the pathophysiologic process of NAFLD was occurred as the result of multiple-hits.5 The rising prevalence and the challenges associated with the treatment of NAFLD have vigorous research in the field over the past 15 years. Studies always tend to focus on better understanding the pathophysiology of NAFLD, as well as the development of therapeutic strategies. However, it is still incompletely understood that critical factors contributing to the progress of NAFLD. Lately gut microbiota has appeared as a crucial environmental aspect concerning in the development of NAFLD.6-9 The gut microbiota has newly been recognized as a main internal metabolic organ, composed of >1014 microorganisms, with a total mass of approximately 0.3% of an individual’s body weight,10 and containing a second genome named as metagenome, which is approximately 450 times genes compared to the host.11 A large mount of evidence suggests that gut dysbiosis can cause intestinal inflammation as well as chronic inflammatory liver disease. To be specific, a close anatomical and functional link between gastrointestinal tract and the liver was naturally existed via the hepatic portal system, which is termed as gut-liver axis. It permits gut bacteria and their metabolic products to transfer into the liver on account of this direct relationship between the two organs and accelerates the progress of NAFLD.12 Fat in diet, couple with other



factors, may give rise to gut dysbiosis, as well as intestinal barrier injury and increased intestinal permeability (“leaky gut”) thus resulting the gut bacteria and bacteria-derived products translocation into the mesenteric portal bloodstream. The main gut bacterial product, possibly related well to NAFLD pathogenesis and an active constituent of endotoxin as well, is lipopolysaccharide (LPS). LPS derived from the gut bacteria is flowed into the liver via the portal circulation. And sequentially Kupffer cells, resident macrophages of liver, are activated through binding to the Toll-like receptor 4 (TLR-4) located on the cell surface and subsequently the inﬂammatory cytokines were induced to produce, which participate in the development of NAFLD.13-16 Beyond what described above, short-chain fatty acids (SCFAs) generated by the gut microbiota through the fermentation of indigestible carbohydrates in the colon mainly include acetate, propionate, butyrate, isobutyrate, valerate and isovalerate, which greatly help to maintain the integrity of intestinal epithelial cell, thereby preventing against NAFLD.17,18 Diammonium glycyrrhizinate (DG), a medicinal form of glycyrrhizic acid as well as a natural and major bioactive pentacyclic triterpenoid glycoside extracted from licorice roots, possesses a comprehensive pharmacological properties such as anti-viral, anti-inflammatory, anti-allergic, anti-oxidant and anti-tumor and so on.19-24 DG is commonly used as one of therapeutic agents for the treatment and control of chronic hepatopathy including NAFLD.25-28 And it also has been proved to alleviate serum liver enzymes and ameliorate pathological damage of liver due to its anti-inflammatory mechanisms of action in liver.27, 29-31 Previous studies have shown that DG was mainly distributed in the liver and intestines after absorbed into the blood.32 In addition, it can significantly alleviate the intestinal mucosa inflammation in mice.30 Therefore, we hypothesize that modulation of gut microbiota and restoring intestinal barrier may play key roles for DG


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protecting against NAFLD. In this current investigation, we reported that DG significantly prevented NAFLD mice induced by high-fat diet (HFD). Interestingly, DG modulated the gut microbiota, restored intestinal barrier impairment, reduced the intestinal low-grade inflammation and finally ameliorated liver steatosis. This study helps us acquire a better understanding of the mechanisms for DG protection against NAFLD.

MATERIALS AND METHODS Animals and drug treatments Four-week old male mice on a C57BL/6J background (20±2 g) were obtained from Beijing HuaFuKang biotechnology Co., LTD. (Beijing, China). Animals were housed under a specific pathogen free (SPF) condition: temperature, 25℃; humidity, 50%; lights on at 6:00 and off at 18:00. All animal experiments were approved by the Animal Care and Use Committee of Tianjin Medical University, Tianjin, P. R. China. A total of 22 mice were fed with a normal diet for 1 week to be adapted to SPF condition and then were randomized into three groups: (1) control group (NCD): mice were treated with normal chow diet for 14 w (n = 6); (2) high-fat diet with DG group (HFD+DG): mice were treated with HFD and injected intraperitoneally with DG on alternate days for 14 w (150 mg/kg, n = 8); (3) high-fat diet with normal saline group (HFD): animals were treated with HFD and intraperitoneally injected with the same volume of vehicle on alternate days for 14 w (0.9% NaCl, 0.3 ml, n = 8). The high-fat diet was comprised of 20% protein kcal, 20% protein kcal carbohydrate, 60% fat kcal and 5.24 kcal/gm. The dose and administration of DG (150 mg/kg, intraperitoneal injection) was selected based on the available studies.26,


33, 34

DG was kindly


provided from Chia Tai Tianqing pharmaceutical Company (Jiangsu, China). Body weight was recorded weekly. Tissue extraction All mice were overnight fasted at the end of 15 weeks feeding period and then killed by CO2 inhalation. Blood samples of suborbital venous plexus were collected into tubes and centrifuged (3500 rpm, 10 min, 4℃). Serum was measured as described below. Liver and intestinal tissues were excised followed by weighting epididymal fat immediately. Related studies have found that most SCFAs are absorbed in the intestine and only a small amount of SCFAs can be detected in fecal samples.35,36 Thus cecal contents were snap-frozen in liquid nitrogen for analysis of SCFAs content. Intestinal specimens and liver tissues were frozen immediately in liquid nitrogen and then stored at −80℃ refrigerator until further protein and RNA extraction. A portion of liver and intestinal tissues were formalin-fixed and paraffin-embedded for further pathological evaluation. Fresh fecal samples were collected for 1 day and frozen in liquid nitrogen for gut microbiota analysis before the end of the experimental period.37 Metabolic indices detection The serum liver enzymes including aspartate aminotransferase (AST) and alanine aminotransferase (ALT), lipid including triglyceride (TG) and total cholesterol (TC) were determined by enzymatic colorimetric method. And these parameters were all determined by the usage of a commercially available kit under the manufacturer's instructions from Beijing Applygen Technologies Inc. (Beijing, China). Histology The 4-µm thick paraffin sections of liver and intestinal tissues were totally stained with


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hematoxylin–eosin (HE) for histological analysis. Liver steatosis stage and colonic inflammation were observed under a light microscope. And five random areas in each tissue section were examined and scored by the same pathologist (YJZ) in a blinded manner. The liver histopathological score was evaluated according to the degree of hepatic steatosis and liver lobular inﬂammation. Hepatic steatosis was graded by the ratio of the fatty hepatocytes occupying hepatic parenchyma. And furthermore, grades according to the ratio described above were divided into 0, 2/3. The inflammation activity scores were composed of 4 parts, which were portal area inflammation (P), lobular inflammation (L), debris-like necrosis (PN) and bridging necrosis (BN). Each part was divided into 1 ~ 4 points according to the degree of liver disease, respectively. Ultimately, the inflammation scoring formula was (P+L+2 PN+2 BN). In addition, colonic histological inflammation was assessed by the degree of crypt damage. Gut microbiota analysis The V4-V5 hypervariable regions of the 16S rRNA gene were subjected to high-throughput sequencing by Shanghai TinyGene Biotechnology, Co., Ltd (Shanghai, China). Fecal DNA from each stool sample was extracted following the manufacturer’s guidelines (QIAamp DNA Stool Mini Kit, Qiagen, Hilden, Germany): 100 mg fecal sample was thoroughly homogenized in 1.4mL ASL buffer. Samples were then mixed with 0.2 g sterile zirconia/silica beads and were subsequently processed on a Tissue Lyser at 30 Hz for 6 minutes. The mixture was heated for 5 minutes at 95°C to lyse fecal bacterial cells and then eluted with elution buffer. Amplification of V4-V5 hypervariable region of 16S rRNA genes was performed using the specific primer with barcode. An equal volume of 1×loading buffer (containing SYB green) and PCR product were mixed and then electrophoresed on a 2% agarose gel. The PCR products were mixed and followed



by purification with GeneJET Gel Extraction Kit (Thermo Scientific). Illumina TruSeq DNA PCR-Free Library Preparation Kit (Illumina, USA) and QIIME software package (Quantitative Insights Into. Microbial Ecology) were used to generate the sequencing libraries and analyze the sequences, respectively.38 Sequences selected at ≥97% similarity were optimized to map into operational taxonomic units (OTUs).39 And QIIME platform was used for further taxonomic analysis based on the OTUs sequences.40 Several metrics of the OTUs rarefaction such as curves of OTU rank, rarefaction and calculated indexes of Shannon, Chao, Simpson and ACE were used for the alpha diversity assessment. Simultaneously, we applied several metrics performed using QIIME including principal component analysis (PCA), principal coordinate analysis (PCoA), non-metric multidimensional scaling (NMDS), heatmap of RDA-identified key OTUs, and unweighted pair group method with arithmetic mean (UPGMA) to analyze the beta diversity. Analysis of short chain fatty acids (SCFAs) in cecal contents The concentrations of SCFAs (acetate, propionate, isobutyrate, butyrate, isovalerate and valerate) in cecal contents were determined using gas chromatography (GC) method. Briefly, cecal contents were diluted with 10 mmol/L NaOH to 3 mL. The feces samples were extracted by vortexing and sonication at 0℃ for 5 min. The pooled extraction was then centrifuged (15 min, 10,000 rpm, 4℃). The supernatant (1 mL) was mixed with chloroform with a ratio of 1:1 to extract liposoluble components, aqueous phase was left after vortexing and sonication. Then 0.7 mL of aqueous phase was used to react with about 1.2 µL HCl for 10 min. Ethyl acetate (1.4 mL) was used to extract the SCFAs in the aqueous phase. The extraction was filtered through 0.22 µm membranes and then analyzed with an Agilent 7890A Series GC. Data were collected and analyzed with Agilent ChemStation software. Different components were identified and quantified


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according to its relative retention time with the reference standards (Sigma-Aldrich, USA) and peak area, respectively. Real-time PCR analysis Total colon tissues RNA were extracted by carrying out the RNeasy mini kit (Qiagen, Carlsbad, CA, USA), and reverse transcription of cDNA was performed using the TIANScript RT kit (TIANGEN, Inc. Beijing, China). Table 1 listed the oligonucleotide primers of the target genes. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was selected as the reference gene due to its uniform expression in each sample and the relative mRNA expression was finally

calculated by 2-∆∆Ct method. Periodic acid Schiff (PAS) staining Deparaffinized colon sections were incubated with 1% Periodic acid solution (Sigma-Aldrich) for 10 minutes. And then were incubated for 40 minutes using Schiff reagent (Sigma-Aldrich) again. After that, Hematoxylin was used for the counterstaining of PAS-stained sections for 2-5 minutes rinsing with extensive PBS solution between each step. Results were observed under light microscopy. Goblet cells in intestinal tissue of each mouse were stained into pink color for evaluating the intestinal barrier. And the number of PAS positive cells was measured by counting the absolute positive staining cells number in total cells number for each mouse. Immunohistochemistry and immunofluorescence The 4-µm thick paraffin-embedded colon sections were deparaffinized and then were boiled for 15 min to unmask antigens. Blocked the tissue sections with 5% goat serum in PBS. For mucin 2 (MUC2) staining, tissue sections were first incubated with a rabbit monoclonal anti-MUC2 (1:250, Santa Cruz Biotechnology, Inc.) overnight at 4°C. Horseradish peroxidase (HRP)-labeled



anti-rabbit secondary antibody was then used for incubating the washed sections for 30 minutes. And it was finally incubated with 3, 3’-diaminobenzidine for color development and observed subsequently using light microscopy. The sections were viewed blindly by the same pathologist (YJZ). And the evaluation of intestinal barrier was determined by the MUC2 positive staining cells number (brown staining) in total cells number for each mouse. For zona occludens-1 (ZO-1) staining, rabbit anti ZO-1 antibody (Invitrogen Corporation, Carlsbad, CA) was first used for incubating the sections at 4°C overnight, and then FITC-labeled anti-rabbit antibody (Cell Signaling Technology) was used for secondary incubation for 1 h at room temperature. DAPI (4, 6-diamidino-2-phenylindole, blue) was lastly added to the slides and subsequently observed the results under the fluorescence microscopy. Statistical analysis Statistical analysis and graph design were performed using SPSS 17.0 (SPSS, Chicago, IL, USA). Data were presented as means ± SD and represented the average of at least three experiments performed in triplicate. One-way ANOVA was applied to check the differences among groups for multiple comparisons with significant difference of P value < 0.05.

RESULTS DG reduced body weight gain and epididymal adipose in HFD-fed mice As anticipated, HFD mice put on more weight than that in the NCD mice. DG Supplementation observably prevented the HFD mice from gaining more weight (Fig. 1 A). After feeding for 2 week, HFD mice and HFD + DG mice gained more weight relative to NCD mice. It was worth noting that the body weight gain in HFD mice was higher than that in the HFD+DG mice, and the increase was gradually higher with the extension of feeding time (Fig. 1 B). Meanwhile, as shown


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in Fig. 1 C, epididymal adipose tissues in mice were remarkably increased by HFD (P

Diammonium Glycyrrhizinate Protects against Non-Alcoholic Fatty

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